Methods for inhibiting stenosis, obstruction, or calcification of a stented heart valve or bioprosthesis

ABSTRACT

Methods for inhibiting stenosis, obstruction and/or calcification of a heart valve following implantation in a vessel having a wall are disclosed. In one aspect the method includes providing a bioprosthetic heart valve mounted on an elastical stent; treating the bioprosthetic heart valve with a tissue fixative; coating the stent and the bioprosthetic valve with a coating composition including one or more therapeutic agents; implanting the bioprosthetic valve into the vessel in a diseased natural valve site; eluting the coating composition from the bioprosthetic valve; and inhibiting stenosis, obstruction and/or calcification of the bioprosthetic heart valve by preventing the attachment of stem cells to the bioprosthetic heart valve, the stem cells circulating external and proximate to the bioprosthetic heart valve by activating nitric oxide production (i) in the circulating stem cells, (ii) in an endothelial cell lining covering the bioprosthetic heart valve tissue, (iii) or both.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/601,236, filed on May 22, 2017, pending; which is acontinuation-in-part of U.S. application Ser. No. 15/193,208, filed onJun. 27, 2016, abandoned; which is divisional of U.S. application Ser.No. 14/263,438 filed on Apr. 28, 2014, abandoned; which is acontinuation-in-part of U.S. application Ser. No. 13/656,925, filed onOct. 22, 2012, abandoned; wherein U.S. application Ser. No. 15/601,236is also a continuation-in-part of U.S. application Ser. No. 14/687,479,filed on Apr. 15, 2015, abandoned; which is a continuation-in-part ofU.S. application Ser. No. 14/263,438 filed on Apr. 28, 2014, abandoned;which is a continuation-in-part of U.S. application Ser. No. 13/656,925,filed on Oct. 22, 2012, abandoned; wherein U.S. application Ser. No.15/601,236 is also a continuation-in-part of U.S. application Ser. No.15/031,532, filed on Apr. 22, 2016, abandoned; which is a 371(c)national stage application claiming priority to International patentapplication Ser. No. PCT/US2014/061745, filed on Oct. 22, 2014, which isa continuation-in-part of U.S. application Ser. No. 14/263,438 filed onApr. 28, 2014, abandoned; which is a continuation-in-part of U.S.application Ser. No. 13/656,925, filed on Oct. 22, 2012, abandoned; theentireties of the foregoing hereby being incorporated by reference.

FIELD OF THE INVENTION

The invention relates to methods for inhibiting stenosis, obstruction,or calcification of heart valves and heart valve prostheses.

BACKGROUND OF THE INVENTION

The heart is a hollow, muscular organ that circulates blood throughoutan organism's body by contracting rhythmically. In mammals, the hearthas four-chambers situated such that the right atrium and ventricle arecompletely separated from the left atrium and ventricle. Normally, bloodflows from systemic veins to the right atrium, and then to the rightventricle from which it is driven to the lungs via the pulmonary artery.Upon return from the lungs, the blood enters the left atrium, and thenflows to the left ventricle from which it is driven into the systemicarteries.

Four main heart valves prevent the backflow of blood during the rhythmiccontractions: the tricuspid, pulmonary, mitral, and aortic valves. Thetricuspid valve separates the right atrium and right ventricle, thepulmonary valve separates the right atrium and pulmonary artery, themitral valve separates the left atrium and left ventricle, and theaortic valve separates the left ventricle and aorta. Generally, patientshaving an abnormality of a heart valve are characterized as havingvalvular heart disease.

One way a heart valve can malfunction is by failing to open properly dueto stenosis, requiring replacement of the valve by surgical ortranscutaneous arterial means. Replacement valves are typicallybioprosthetic valves made from valves of other animals, such as pig orcow. Unfortunately, over time, the replacement valves themselves aresusceptible to problems such as stenosis, obstruction and calcification.

For years cardiac calcification was thought to be a passive degenerativephenomenon. Rajamannan's work in U.S. Pat. Appln. No. 2002/0159983 firstattempted, unsuccessfully, to address the issue of stenosis andcalcification of diseased heart valves. Rajamannan discloses a methodfor inhibiting stenosis, obstruction or calcification of heart valvetissue having live, non-fixed heart valve cells that contain anexogenous nucleic acid that encodes or activates endothelial nitricoxide synthase—a polypeptide having nitric oxide synthase activity. Itwas thought at the time that nitric oxide would stop the calcificationof the live tissue before it was processed using glutaraldehyde.Rajamannan used a virus (e.g. retrovirus, adenovirus, or herpes virus)to introduce the exogenous nucleic acid (eNOS) by injection into a livecell of the heart valve (human or porcine—in vivo or in vitro) such thatthe polypeptide—endothelial nitric oxide synthase—is expressed. It wasthought that the cell would make excessive amount of nitric oxidesynthase that would stop calcification. Rajamannan also disclosesadministering an inhibitor of hydroxymethylglutaryl coA reductaseactivity, such as pravastatin, atorvastatin, simvastatin or lovastatinorally to the mammal and/or bathing the cells with bovine serumcontaining the foregoing. After the foregoing treatment, the human orporcine heart valve was fixed with glutaraldehyde. As noted above, thistechnique was unsuccessful.

What is needed are new methods and systems that inhibit the formation ofstenosis, obstruction, and/or calcification of heart valves andbioprosthetic heart valves.

BRIEF SUMMARY OF THE INVENTION

The heart valve and method in accordance with the invention addressesthe shortcomings with conventional bioprosthetic, surgical andmechanical heart valves.

In one aspect the method for inhibiting stenosis, obstruction and/orcalcification of a heart valve following implantation in a vessel havinga wall includes providing a bioprosthetic heart valve comprising tissuehaving one or more cusps, the bioprosthetic heart valve mounted on anelastical stent for replacement of a natural diseased valve; treatingthe bioprosthetic heart valve with a tissue fixative; coating the stent,one or more cusps, or both with a coating composition comprising one ormore therapeutic agents; implanting the bioprosthetic valve into thevessel in a diseased natural valve site; eluting the coating compositionfrom the elastical stent, one or more cusps, or both; inhibitingstenosis, obstruction and/or calcification of the bioprosthetic heartvalve by preventing the attachment of stem cells to the bioprostheticheart valve, the stem cells circulating external and proximate to thebioprosthetic heart valve, elastical stent or both by activating nitricoxide production (i) in the circulating stem cells, (ii) in anendothelial cell lining covering the bioprosthetic heart valve tissue,(iii) or both.

In another aspect of the invention, the method the stem cells are cKitpositive stem cells SCA1 cells, COP cells, mesenchymal stem cells andcombinations of the foregoing.

In another aspect of the invention, the method includes treating thebioprosthetic heart valve tissue with an eNOS activator selected fromAtorvastatin, Rosuvastatin, Pravastatin, Mevastatin, Fluvastatin,Simvastatin, Lovastatin, L-Arginine, citrulline, NADPH, acetylcholine,histamine, arginine vasopressin, norepinephrine, epinephrine,bradykinin, adenosine di,triphosphate, 5-Hydroxytrptamine, thrombin,insulin, glucocorticoids, salicylates, L-NMMA, L-NAME, nitroglycerine,isosorbide dinitrate, isosorbide 5-mononitrate, amyl nitrite,nicorandil, tetrahydrobiopterin, Zetia and combinations of theforegoing.

In another aspect of the invention, the coating composition includes oneor more of (i) an anti-proliferative agent; (ii) an inhibitor ofextracellular production; (iii) an inhibitor of osteoblastogenesis; and(iv) combinations of the foregoing for inhibiting bone formation in anosteoblast cell originating from the stem cells.

In another aspect of the invention the anti-proliferative agent ispaclitaxel, sirolimus, biolimus, everolimus and combinations of theforegoing.

In another aspect of the invention the inhibitor of extracellularproduction is an anti-farnysltransferase inhibitor, ananti-palmitoylation inhibitor or both.

In another aspect of the invention the anti-famysltransferase inhibitorand/or the anti-palmitoylation inhibitor include Lonafarnib, ZarnestraR115777, FTI SCH66336, STI571,FLT-3 Inhibitor, Proteasome Inhibitor,MAPK Inhibitor, BMS-214662, Type I non-lipid inhibitors of proteinpalmitoylation, farnesyl-peptide palmitoylation or Type 2 inhibitorsmyristoyl-peptide palmitoylation, palmitoylation acyltransferaseinhibitors, lipid based palmitoylation inhibitors including2-bromopalmitoyl (2BP), tunicamycin, cerulnin and combinations of theforegoing.

In another aspect of the invention the inhibitor of osteoblastogenesisincludes anti-osteoporotic agents such as bisphosphonate drugs includingAlendronate, Risedronate, Zoledronic acid, Etidronate, Ibandronate,Pamidronate, Tiludronate, Denosumab antibody, Calcitonin-Calcimare,Miacalcin, Forteo teriparatide, raloxfine (Evista) and combinations ofthe foregoing.

In another aspect of the invention an oral dosage of eNOS activators areadministered to a patient. The oral dosages include 10 mg to 80 mg perday of Atorvastatin; 10 mg to 40 mg Simvastatin per day; 5 mg to 40 mgRosuvastatin per day; 10 mg to 40 mg Pravastatin per day; 1 mg to 4 mgPitavastatin per day; 10 mg Zetia per day and combinations of theforegoing.

In another aspect of the invention an oral dosage of theanti-famysltransferase inhibitor, the anti-palmitoylation inhibitor orboth is administered to a patient. The dosages may include Lonafarnib ina dosage of 115 mg/m2 dose with a range from 115 mg/m2 to 150 mg/m2, incombination with an effective amount of Zetia of 10 mg.

In another aspect of the invention, an effective amount of a PCSK9inhibitor is administered to a patient by intramuscular or subcutaneousinjection. An initial dose of the PCSK9 inhibitor may be from 0.25 mg/kgto 1.5 mg/kg; from 0.5 mg/kg to 1 mg/kg.

In another aspect of the invention an effective amount of the PCSK9inhibitor is Alirocumab 75-150 mg every 2 to 4 weeks, Evolocumab 140 mgevery 2 weeks or monthly and combinations of the foregoing.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the invention, and to show how the samemay be carried into effect, reference will now be made, by way ofexample, to the accompanying drawings, in which:

FIG. 1 is an illustration of the commercial process of obtaining a heartvalve from a cow or pig and fixing it with glutaraldehyde to make thetissue inert and placing the tissue into a stented valve or surgicalreplacement valve.

FIG. 2A is a perspective view of a bioprosthetic surgical heart valvewith a sewing ring covered with tissue.

FIG. 2B is a top view of a mechanical heart valve having a Gortexcovering on the sewing ring.

FIG. 2C is a perspective view of a transcutaneous, stented bioprostheticheart valve.

FIG. 3 illustrates the cellular origins of cardiac calcification and thethree stages of bone formation on heart valves.

FIG. 4 depicts light microscopy slides of implanted valves from thethree rabbit treatment groups with the left column, control diet; middlecolumn, cholesterol diet; and right column, cholesterol diet plusatorvastatin. (All frames 20× magnification, with a 100× magnificationin the right hand lower corner) Panel A. Masson Trichrome stain. PanelC. Proliferating cell nuclear antigen stain. Panel D. Osteopontin stain.

FIG. 5 depicts the Semi-Quantitative RT-PCR of the implanted valves fromthe three rabbit treatment groups; RT-PCR using the total RNA from thebioprosthetic valves for cKit(731 bp), Cyclin(151 bp), Osteopontin (OP)(102 bp), Sox9(170 bp), Cbfa-1 (289 bp), and GAPDH (451 bp).

FIG. 6 illustrates the mechanism for bioprosthetic valve calcification.

FIG. 7 is a table setting forth the real-time polymerase chain reaction(RTPCR) results from the pericardial valves removed from the rabbitstudies.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for inhibiting stenosis, obstruction,and/or calcification of a valve leaflets and valve tissue in a stentedbioprosthetic heart valve 11 or a surgical replacement valve 10 with orwithout a sewing ring 12, following fixation of the valve and subsequentimplantation of the valve in a patient in need thereof. The inventionalso provides a method for inhibiting stenosis, obstruction, and/orcalcification of a mechanical heart valve 14 having a Gortex lining 16surrounding the sewing ring 18 of the mechanical heart valve.

As used herein, the term “stenosis” refers to the narrowing of a heartvalve that could block or obstruct blood flow from the heart and cause aback-up of flow and pressure in the heart. Valve stenosis may resultfrom various causes, including, but not limited to, scarring due todisease, such as rheumatic fever; progressive calcification; progressivewear and tear; among others. This is important not for the stentedtreatment but for the valve—is this flowing well with the rest of thepatent.

As used herein, the term “valve” may refer to any of the four main heartvalves that prevent the backflow of blood during the rhythmiccontractions. The four main heart valves are the tricuspid, pulmonary,mitral, and aortic valves. The tricuspid valve separates the rightatrium and right ventricle, the pulmonary valve separates the rightatrium and pulmonary artery, the mitral valve separates the left atriumand left ventricle, and the aortic valve separates the left ventricleand aorta. Thus, in one aspect, the bioprosthetic valve and the diseasedvalve may be an aortic valve, pulmonary valve, tricuspid valve, ormitral valve.

As used herein, the term “bioprosthetic valve” is a stented tissue heartvalve and may refer to a device used to replace or supplement a heartvalve that is defective, malfunctioning, or missing. Examples ofbioprosthetic valve prostheses include, but are not limited to, EdwardsSapien 1 Transcatheter Heart Valve, Edwards Sapien 2 Transcatheter HeartValve, Edwards Sapien 3 Transcatheter Heart Valve, and all othervariations of the Edwards Sapien Transcatheter valves, including but notlimited to Edwards Sapien XT Transcatheter Heart Valve, BostonScientific Lotus Edge Transcatheter Heart Valve and all variations ofthe Lotus Valve, Medtronic Core Valve Transcatheter Valve and MedtronicCore Valve Evolut R Transcatheter Valve, Melody Transcatheter PulmonaryValve Therapy and all variations thereof.

Generally, bioprosthetic comprise a tissue valve having one or morecusps and the valve is mounted on a frame or stent, both of which aretypically elastical. As used herein, the term “elastical” means that thedevice is capable of flexing, collapsing, expanding, or a combinationthereof. The cusps of the valve are generally made from tissue ofmammals such as, without limitation, pigs (porcine), cows (bovine),horses, sheep, goats, monkeys, and humans.

As used herein a “surgical heart valve” is one harvested from pigs orcows comprising only tissue and a sewing ring. The surgical heart valveis typically stentless. Non-limiting examples of surgical heart valvesinclude ATS 3F Aortic Bioprosthesis, Carpentier-Edwards PERIMOUNT MagnaEase Aortic Heart Valve, Carpentier-Edwards PERIMOUNT Magna Aortic HeartValve, Carpentier-Edwards PERIMOUNT Magna Mitral Heart Valve,Carpentier-Edwards PERIMOUNT Aortic Heart Valve, Carpentier-EdwardsPERIMOUNT Plus Mitral Heart Valve, Carpentier-Edwards PERIMOUNT TheonAortic Heart Valve, Carpentier-Edwards PERIMOUNT Theon MitralReplacement System, Carpentier-Edwards Aortic Porcine Bioprosthesis,Carpentier-Edwards Duraflex Low Pressure Porcine Mitral Bioprosthesis,Carpentier-Edwards Duraflex mitral bioprosthesis (porcine),Carpentier-Edwards Mitral Porcine Bioprosthesis, Carpentier-EdwardsS.A.V. Aortic Porcine Bioprosthesis, Edwards Prima Plus StentlessBioprosthesis, Medtronic, Freestyle® Aortic Root Bioprosthesis, Hancock®II Stented Bioprosthesis, Hancock II Ultra® Bioprosthesis, Mosaic®Bioprosthesic, Mosaic Ultra® Bioprosthesis, St. Jude Medical, Biocor®,Biocor™ Supra, Biocor® Pericardia, Biocor™ Stentless, Epic™ , EpicSupra™, Toronto Stentless Porcine Valve (SPVO), Toronto SPV II®,Trifecta, Sorin Group, Mitroflow Aortic Pericardial Valve®, Cryolife,Cryolife aortic Valve® Cryolife pulmonic Valve®, Cryolife-O'Brienstentless aortic xenograft Valve®Contegra Pulmonary Valve Conduit, EpicStented Tissue Valve with Linx AC Technology, Aortic and Mitral Valves;Trifecta Valve with Guide Technology, Aortic and Mitral Valves.

As used herein a “mechanical heart valve” is one which is typicallymanufactured from a biocompatible material such as pyrolytic carbon.Non-limiting examples of mechanical heart valves include the SJM RegentHeart Valve, Mitral and Aortic Position; and the Starr-Edwards Valve,Mitral and Aortic Valves; Smeloff Cutler Valve, Mitral and AorticValves; Caged Ball Valve, Mitral and Aortic Valves; tilting disk valve,Mitral and Aortic Valves,; bileaflet valve, Mitral and Aortic Valves;Bjork-Shiley Valve, Mitral and Aortic Valves; Medtronic Hall HeartValve, Mitral and Aortic Valves, Annuloplasty Rings including the PhysioI and Physio II, Duran AnCore Ring and Band, CG Future Composite Ringand Band, Profile 3D Ring, Open Pivot Mechanical Heart Valve, MedtroincOpen Pivot Aortic Valved Graft, Simulus Adjustible Annuloplasty System,Simulus Flexible Annuloplasty System, Simulus Semi-Rigid AnnuloplastySystem, Amplatzer Devices including but not limited to Patent ForamenOvale, Ventricular Septal Closure Device, Atrial Septal Defect Device,Patent Ductus Arteriosus Device, and CryoLife On-X Valves, including butnot limited to Aortic Heart Valve with Conform-A Sewing Ring, AorticHeart Valve with Anatomic Sewing Ring, Aortic Heart Valve with StandardSewing Ring, Mitral Heart Valve with Standard Sewing Ring, Mitral HeartValve with Conform-X sewing Ring, Ascending Aortic Prosthesis.

Referring now to FIG. 1 two types of materials used for bioprostheticheart valves, bovine pericardial and/or porcine aortic valve cusps, insurgical valve replacement versus transcutaneous valve replacement ofnative diseased valves are shown. The structural elements of thebiomaterial and the modification of the tissue prior to placement in thesewing ring and or the surgical stent is a routine tissue preparationpretreatment called fixation by using aldehyde solutions to cause across linking of the tissue making it non-viable or inert, as shown inFIG. 1. “Fixation” of the tissue is performed using variousconcentrations of glutaraldehyde or formaldehyde. This pretreatment isperformed to improve tissue durability using glutaraldehyde orformaldehyde as cross-linking agents.

After the valve leaflet, either bovine pericardial and or porcineaortic, is treated with glutaraldehyde or formaldehyde fixation andattached to the sewing ring and/or stent it is then placed in the humanbody to replace the diseased valve in one of two ways. The surgicalapproach involves opening the chest cavity to replace the diseasedvalve. The transcutaneous approach involves positioning thebioprosthetic valve on a catheter and threading it through an arteryuntil the bioprosthetic valve reaches the diseased valve site where thediseased valve is replaced with the bioprosthetic valve using techniquesknown to those of skill in the art. A transapical approach may also beused wherein the bioprosthetic valve is introduced into the diseasedvalve site through the apex of the heart using techniques known to thoseof skill in the art.

Referring now to FIGS. 2A-2C, the three different types of heart valvesare illustrated. FIG. 2A depicts a bioprosthetic surgical heart valve 10having three leaflets 20, 21, 22 and a sewing ring 12 covered by tissue.FIG. 2B depicts a mechanical heart valve 14 having two mechanicalleaflets 23, 24. A mechanical heart valve is placed surgically using theopen-chest procedure. While mechanical heart valves 14 do not use tissuefor the leaflets, the valve structure itself 14, 23, 24 as well as theGortex lining 16 covering the sewing ring 18 of mechanical heart valvesmay also be at risk for calcification. FIG. 2C depicts a bioprostheticheart valve 11 mounted on stent 26. Stented bioprosthetic valves aretypically placed transcutaneously through an artery and may also beplaced transapically.

In order to understand the method in accordance with the invention ofinhibiting calcification, it is important to understand the mechanism ofcalcification, which is illustrated in FIGS. 3 and 6. As the valve isplaced in the heart, either aortic, pulmonic, tricuspid and or mitralposition in the heart, the heart produces an endothelial lining over theentire valve surface area including the leaflets, stent and the sewingring during the several weeks following implantation and in the case ofmechanical valves over the Gortex. Over time, exposure to stresselements such as high cholesterol, high blood pressure (hypertension),diabetes, smoking, and high levels of C-reactive protein cause theendothelial lining to decrease the eNOS function of the endothelialnitric oxide synthase gene and decrease the production of nitric oxideby the endothelial lining. Less nitric oxide production results in (i)the release of Wnt from the endothelial cells and (ii) the endothelialcells becoming “sticky” due to the decrease in production of nitricoxide. The release of Wnt in turn activates the Wnt cascade incirculating stem cells. Because the endothelial cells are “sticky” stemcells, cKIT positive cells, SCA1, circulating osteogenic precursorcells, mesenenchymal circulating stem cells, and combinations of theforegoing, attach to the abnormal endothelial lining that has grown onthe valve. The release of Wnt and the activation of the Wnt cascade inthe foregoing cells triggers the three stages of bone Ruination, namelycell proliferation, extracellular matrix production of cartilagespecific proteins and osteoblastogenesis wherein the stem cells becomesan osteoblast cell that deposits bone matrix (calcium and phosphate)into the cartilage scaffold where the calcium and phosphate bind to thebone matrix. This bone matrix or calcification covers the bioprostheticvalve like a “shell,” which is calcification and represents boneformation.

The present inventor tested the hypothesis that bioprosthetic valvecalcification is a stem cell mediated atherosclerotic process. A modelof prosthetic valve calcification for stem cell markers were tested.Normal bioprosthetic valves were implanted subcutaneously in rabbits(n=10) control, (n=10) 0.5% Cholesterol diet and (n=10) 0.5%Cholesterol+Atorvastatin for three months to analyze for atheroscleroticbone formation. The bioprosthetic valve tissue explanted from therabbits fed the cholesterol diets demonstrated severe atherosclerosiswith islands of stem cell positive for ckit, macrophage, and osteopontinexpression. Control valves demonstrated a mild increase in the markers.Atorvastatin treated valves had no evidence of stem markers oratherosclerosis (p<0.05). Bioprosthetic valve calcification is amesenchymal ckit mediated atherosclerotic calcification process, whichis attenuated by atorvastatin. These experimental data have implicationsfor future therapy of patients with bioprosthetic valve to slow theprogression of valve calcification.

Calcific aortic stenosis is the most common indication for surgicalvalve replacement in the United States and Europe. Currently, mechanicalversus bioprosthetic heart valves are the two options for valvereplacement. The choice of valve depends on patient characteristics atthe time of surgery. Bioprosthetic heart valves have decreased risk ofthrombosis, therefore decreasing the need for anticoagulation.Therefore, these are the valves of choice, in patients who are olderthan 75 years of age or who have contraindications to long-termanticoagulation, despite their limited long-term durability. A recentstudy demonstrated the expression of bone proteins in human aortic valveallografts setting the foundation for this disease process. It isestimated that 20-30% of implanted bioprosthetic heart valves will havesome degree of hemodynamic dysfunction at 10 years. For years, themechanisms of valve degeneration, was thought to be due to a passiveprocess. However, recent studies have demonstrated risk factors forbioprosthetic valve calcification that are similar to vascularatherosclerosis. Furthermore, recent pathologic studies, have clearlyshown that an inflammatory reaction develops in these calcifyingbioprostheses which includes lipid deposits, inflammatory cellinfiltration, and bone matrix proteins expression. These findingsparallel a similar histopathologic lesion found in native calcificaortic valve disease.

To study the mechanism by which bioprosthetic heart valves calcify, thisstudy tested a hypothesis that bioprosthetic valve calcification occurssecondary to a stem cell mediated atherosclerotic process. Ifatherosclerotic risk factors are independently associated with thedevelopment of bioprosthetic valve deterioration, then experimentalmodels of atherosclerosis may provide a method to test the developmentof bioprosthetic valvular heart disease.

This study evaluated surgically implanted bovine pericardial valves in arabbit model of hypercholesterolemia to determine if a similaratherosclerotic lesion develops along the surface of the implantedvalve. To test the hypothesis that mesenchymal cKit positive stem cellshome to the valves in the presence of inflammation. Finally, the studysought to determine if atorvastatin attenuates this atheroscleroticprocess in the experimental model.

The implanted valve leaflets from the control animals appeared to have amild amount of cellular infiltration along the surface of the leaflet asdemonstrated by Masson Trichrome stain FIG. 4, Panel A1. The high powermagnification demonstrates the demarcation between the leaflet andcellular infiltrate that develops along the leaflet surface. There was asmall amount of cKit positive staining cells in control bioprostheticvalves FIG. 4, Panel B1, as well as a mild amount of proliferating cellsexpressing osteopontin, as shown in FIG. 4, Panels C1 and D1. Incontrast, in the valve tissue from the cholesterol-fed rabbits FIG. 4,Panels A2, B2, C2, D2, there was marked cellular inflammatory infiltrateas shown in the Masson Trichrome, the cellular infiltrate express cKit,PCNA and OPN as shown in the high resolution field in the bottom lefthand corner, with the blue staining cells. Finally, as measured bysemi-quantitative visual analysis, at the time of explant and under thelight microscopy, the atherosclerotic burden increased four-fold withthe cholesterol treatment. The implanted leaflets in the atorvastatintreated rabbits demonstrated a marked decrease in the amount ofatherosclerotic cellular infiltrate, proliferation, cKit and osteopontinexpression FIG. 4, Panels A3, B3, C3, D3. The immunohistochemistry andMasson Trichrome demonstrate a dramatic improvement in the leaflets ofthe atorvastatin treated animals compared to the leaflets in thecholesterol-fed and control animals. Morphometric analysis demonstrateda 4-fold increase in the atherosclerotic burden as compared to theatorvastatin treated valves which attenuated the atherosclerosissignificantly (p<0.05), the control had a 2-fold increase ininflammation secondary to the effect of implanting the valve in therabbit inducing an inflammatory response.

FIG. 5, demonstrates the RNA gene expression for the control,cholesterol and cholesterol plus atorvastatin treated rabbits. There wasan increase in the Sox9 osteoblast transcription factor, Cyclin, andcKit in the leaflets of the cholesterol-fed animals as compared to thecontrol with Cyclin and cKit significantly increased in the cholesterolover control(p<0.05) with atorvastatin attenuating cKit, Cyclin and Sox9as compared to the cholesterol group (p<0.05). There was no expressionof Cbfa1 and no difference in the osteopontin gene expression in allthree different treatment groups. FIG. 7, is the RTPCR data from therabbit study.

The serum cholesterol levels were significantly higher in thecholesterol fed compared to control animals (1846.0±525.3 mg/dL vs.18.0±7 mg/dL, p<0.05). Atorvastatin treated rabbits manifested lowercholesterol levels than the rabbits receiving the cholesterol diet alone(824.0±152.1mg/d1, p<0.05). There was an increase in hsCRP serum levelsin the cholesterol fed compared to control rabbits (13.6±19.7 mg/dl vs.0.24±0.1 mg/dL, p<0.05), which was reduced by atorvastatin (7.8±8.7mg/dL, p<0.05) as per FIG. 7.

Recent epidemiological studies have revealed that the risk factors forbioprosthetic valve calcification, namely male gender, smoking, andelevated serum cholesterol, are similar to the risk factors associatedwith vascular atherosclerosis. This data demonstrate that experimentalhypercholesterolemia produces biochemical and morphologic evidence ofatherosclerotic changes along the surface of bioprosthetic pericardialvalve tissue that are similar to the changes found in the early stagesof aortic valve disease.

Specifically, there is an atherosclerotic lesion along the valve surfaceof animals fed a high-cholesterol diet, and attenuation of this processwith atorvastatin. In this model, the hypercholesterolemic bioprostheticvalve not only developed an atherosclerotic lesion that isproliferative, but a lesion that expresses increased levels of bonematrix proteins, by immunohistochemistry and osteoblast bone marker,Sox9. The hsCRP levels are also increased in this model of experimentalhypercholesterolemia, indicating an inflammatory environment for thehoming of mesenchymal stem cells to the implanted leaflets. In therabbit subcutaneous model, confirmation of an increase in Sox9expression in bioprosthetic tissue removed from the hypercholesterolemicrabbits with associated atherosclerosis.

The finding of cKit and other mesenchymal expression, in theseatherosclerotic bioprosthetic valves provides a mechanism ,by whichcalcification can develop as shown in the mechanism. Referring to FIG.6, the mechanism for bioprosthetic valve calcification is illustrated.In the presence of elevated cholesterol, hypertension, smoking,diabetes, and/or the process of abnormal oxidative stress, circulatingstem cells home to a normal bioprosthetic heart valve and attach to thevalve leaflet in the presence of cellular inflammation. The inflammationcauses the circulating cKit positive cells, any and all types ofosteogenic precursor cells, mesenchymal cells, seal positive cells, toattach to the ventricular and aortic surface causing calcification onboth sides of the valve leaflet and/or stent and/or sewing ring. In thepresence of risk factors, primarily hyperlipidemia which causes abnormaloxidative stress and abnormal endothelial nitric oxide synthase functionin the newly formed endothelial lining cells lining around the implantedheart valve. The circulating cKit positive cells, circulating osteogenicprecursor cells, mesenchymal cells, seal positive cells, “stick” to thenewly formed endothelial lining along the implanted valve. These varioustypes of stem cells have the ability to differentiate along theosteogenic pathway to form bone on the valve leaflet causingbioprosthetic valve leaflet deterioration. This differentiation processor a cell transformation process from one cell type to another occursonce Wnt is secreted from the newly formed endothelial lining cell,which surrounds the implanted valve. As the stem cells stick onto thenewly formed endothelial lining in the presence of elevated lipids, andthe close proximity of the stem cells sticking to the endothelial liningallows for the secreted Wnt to bind to the Lrp5 receptor which islocated on the cell membrane of the “attached stem cell.” Once theLrp5/Wnt bind then the stem cell differentiates or transforms to a boneforming cell. An example of a compound to stop the cells from attachingto the valve, is the use of Atorvastatin as tested in the animal modelto reduce inflammation to reduce the number of stem cells attaching tothe valve leaflet, and slow the progression of the calcification. In thepresence of hypercholesterolemia, cKit positive cells attach to thebioprosthetic valve and start to differentiate via an osteogenic geneprogram, first Sox9 upregulation in the low pressure model of thesubcutaneous implant in vivo and then, Cbfal upregulation in the highpressure model of the ex vivo human explanted valves from surgery. Therest of the cells are native myofibroblasts differentiating into anosteoblast phenotype as implicated in other studies of aortic valvedisease. In this model, as the environment of hypercholesterolemiainduces inflammation, mesenchymal stem cells mobilize and home to theimplanted bioprosthetic valve. These cells have the potential todifferentiate along the osteogenic pathway as indicated in previousstudies.

The foregoing findings demonstrate in an in vivo experimental model thathypercholesterolemia expresses cKit positive cells, atherosclerosis, andthe osteogenic gene expression in pericardial bioprosthetic valveleaflets that may be a critical mechanism in bioprosthetic valvecalcification.

To address the foregoing mechanism of valve calcification, the presentinventor has invented a way to inhibit the stenosis, obstruction and/orcalcification of the valves by inhibiting (i) three stages of boneformation, i.e. proliferation of circulating ckit and Seal positive stemcells, osteogenic cells, and mesenchymal stem cells in areas externaland proximate to the bioprosthetic or mechanical valve, preventingextracellular matrix production of cartilage specific proteins, andosteoblastogenesis or deposition of bone matrix into the cartilagescaffold; and (ii) the attachment of the foregoing cells to the heartvalve and/or Gortex lining on mechanical heart valves. The foregoingprevention is accomplished by activating nitric oxide in the endothelialcells themselves.

To activate nitric oxide in the endothelial cells, the heart valveleaflets are coated, post-fixation with glutaraldehyde, with a coatingcomposition including one or more therapeutic agents, which adhere tothe heart valve leaflets, stent and/or Gortex in the case of amechanical heart valve. The coating composition may be deposited on bothsides of the valve leaflets, the stent and/or sewing ring to which thebioprosthetic valve is secured, or the Gortex lining covering the sewingring in the case of a mechanical valve, thereby inhibiting stenosis,obstruction, or calcification of the valve prosthesis followingimplantation in a patient in need thereof.

The coating compositions may be prepared by dissolving or suspending apolymer and therapeutic agent in a solvent. Suitable solvents that maybe used to prepare the coating compositions include those that maydissolve or suspend the polymer and therapeutic agent in solution.Examples of suitable solvents include, but are not limited to,tetrahydrofuran, methylethylketone (MEK), chloroform, toluene, acetone,isooctane, 1,1,1, trichloroethane, dichloromethane, isopropanol, andmixtures thereof. However, solvents are not required in many cases.

The coating compositions may be applied by any method to the surface ofthe elastical stent portion of the valve prosthesis or bioprostheses andsewing ring, known by one skilled in the art. Suitable methods forapplying the coating compositions to the surface of the elastical stentportion of the valve prosthesis include, but are not limited to,spray-coating, painting, rolling, electrostatic deposition, ink jetcoating, and a batch process such as air suspension, pan-coating orultrasonic mist spraying, or a combination thereof.

After the coating composition has been applied, it may be cured. As usedherein, “curing” may refer to the process of converting any polymericmaterial into the finished or useful state by the application of heat,vacuum, and/or chemical agents, which application inducesphysico-chemical changes. The applicable time and temperature for curingare determined by the particular polymer involved and particulartherapeutic agent used as known by one skilled in the art. Also, afterthe elastical stent is coated, it may be sterilized by methods ofsterilization as known in the art (see, e.g., Guidance for Industry andFDA Staff—Non-Clinical Engineering Tests and Recommended Labeling forIntravascular Stents and Associated Delivery Systemshttp://www.fda.gov/medicaldevices/deviceregulationandguidance/guidancedocuments/ucm071863.htmand U.S. Pat. No. 7,998,404 entitled “Reduced temperature sterilizationof stents.”

The coating composition may include from one to four categories oftherapeutic agents. The first category may be an eNOS activator; thesecond category an anti-proliferative agent to inhibit cellproliferation; the third category inhibits extracellular matrixproduction; and the fourth category inhibits the osteoblastogenesis.

To inhibit the attachment of the stem cells onto the “sticky” valvestructure the goal is to upregulate the endothelial nitric oxide in thenewly grown endothelial lining on the valve structure and in thecirculating stem cells to prevent attachment of the stem cells to thevalve structure. Agents that upregulate the production of nitric oxide(eNOS activators) may be selected from Atorvastatin, Rosuvastatin,Pravastatin, Mevastatin, Fluvastatin, Simvastatin, Lovastatin,L-Arginine, citrulline, NADPH, acetylcholine, histamine, argininevasopressin, norepinephrine, epinephrine, bradykinin, adenosinedi,triphosphate, 5-Hydroxytrptamine, thrombin, insulin, glucocorticoids,salicylates, L-NMMA, L-NAME, nitroglycerine, isosorbide dinitrate,isosorbide 5-mononitrate, amyl nitrite, nicorandil, tetrahydrobiopterinand combinations of the foregoing.

In addition, agents that upregulate nitric oxide and lipid loweringdrugs may be given orally in varying dosages depending on a patient'sweight. Such agents include Atorvastatin (10 to 80 mg per day dependingon the LDL levels in the patient), Rosuvastatin (5-40 mg a day),Pravastatin (10-40 mg a day), Mevastatin (5-40 mg a day), Fluvastatin(5-40 mg a day), Simvastatin (5-40 mg a day), Lovastatin (5-40 mg aday), Zetia (10 mg a day) and combinations of the foregoing.

A PCSK9 inhibitor may be given by intramuscular injection to the patientin a dosage that correlates with the patient's weight. The initial doseof a PCSK9 inhibitor may be about 0.25 mg/kg, about 0.5 mg/kg, about 1mg/kg or about 1.5 mg/kg, and the initial dose and the first subsequentdose and additional subsequent doses may be separated from each other intime by about one week. PCSK9 inhibitors and their dosages includeAlirocumab in an amount of from 75-150 mg every 2 to 4 weeks andEvolocumab 140 mg every 2 weeks, may be given by subcutaneous injection,bimonthly or monthly and combinations of the foregoing. PCSK9 is aregulator of plasma lipoprotein cholesterol (LDL-C) and an agent that iseffective in risk reduction in coronary artery disease. The proproteinconvertase subtilisin/kexin type 9 (PCSK9) protein regulates theactivity of low-density lipoprotein (LDL) receptors, which are locatedalong the endothelium and in the liver of the patient. Inhibition ofPCSK9 with a monoclonal antibody results in increased cycling of LDLreceptors and increased clearance of LDL cholesterol (LDL-C). Highlyexpressed in the liver, PCSK9 is secreted after the autocatalyticcleavage of the prodomain, which remains non-covalently associated withthe catalytic domain. The catalytic domain of PCSK9 binds to theepidermal growth factor-like repeat A (EGF-A) domain of low densitylipoprotein receptor (LDLR). Both functionalities of PCSK9 are requiredfor targeting the LDLR-PCSK9 complex for lysosomal degradation andlowering LDL-C, which is in agreement with mutations in both domainslinked to loss-of-function and gain-of-function.

Atorvastatin may be given orally in the range of 10 mg to 80 mg,Simvastatin in the range of 10 mg to 40 mg, Rosuvastatin 5 mg to 40 mg,Pravastatin 10 mg to 80 mg, Pitavastatin 1 mg to 4 mg.

It is equally important to include in the coating compositiontherapeutic agents that that inhibit the three stages of osteogenic boneformation. Agents selected from paclitaxel, sirolimus, biolimus,everolimus, tacrolimius and combinations of the foregoing may beincluded in the coating composition to inhibit cell proliferation.

The coating composition may also include inhibitors of extracellularmatrix production such as an anti-farnysltransferase inhibitor (FTI) andan anti-palmitoylation inhibitor. Such agents may include Lonafarnib,Zarnestra R115777, FTI SCH66336, STI571,FLT-3 Inhibitor, ProteasomeInhibitor, MAPK Inhibitor, BMS-214662, Type I non-lipid inhibitors ofprotein palmitoylation, farnesyl-peptide palmitoylation or Type 2inhibitors myristoyl-peptide palmitoylation, palmitoylationacyltransferase inhibitors, lipid based palmitoylation inhibitorsincluding 2-bromopalmitoyl (2BP), tunicamycin, cerulnin and combinationsof the foregoing.

FTI inhibitors anti-palmitoylation inhibitors may also be given orally.Lonafarnib may be given in a dosage of 115 mg/m2 dose with a range from115 mg/m2 to 150 mg/m2, in combination with an effective amount of Zetiaof 10 mg.

The coating composition may also include anti-osteoporotic agents suchas bisphosphonate drugs including Alendronate (5-70 mg orally dependingon the patient's clinical status), Risedronate (5-150 mg orally a daydepending on the patient's clinical status), Zoledronic acid (5 mg IVinfusion over 15 min once a year), Etidronate (20 mg /kg per day for onemonth after placement of the valve), Ibandronate (150 mg orally once amonth), Pamidronate (60-90 IV infusion one time dose), Tiludronate (400mg orally once a day for 3 months), Denosumab antibody (60 mgsubcutaneous injection every 6 months), Calcitonin-Calcimare (60 mgorally each day), Miacalcin (60 mg orally each day), Forteo Teriparatide(60 mg orally each day), Raloxfine (Evista) (60 mg orally each day) andcombinations of the foregoing. These agents will inhibit the final boneformation stage by inhibiting bone formation in the implanted valve.

The foregoing agents that treat the third phase of bone formation mayalso be given to the patient orally or by intramuscular injection in adosages that corresponds with the patient's weight.

EXAMPLE I

A bioprosthetic heart valve is coated with a coating compositionincluding an eNOS activator, Atorvastatin, an anti-proliferative agent,paclitaxel; an inhibitor of extracellular production, ananti-farnysltrasferase inhibitor; and an inhibitor ofosteoblastogenesis, a bisphosphonate durg. The bioprosthetic heart valveis implanted in a 75 year old female patient. The patient also receiveslipid lowering therapy including atorvastatin 80 mg by mouth each dayand a PCSK9 inhibitor once a month in an amount of 75 mg by subcutaneousinjection to lower her LDL cholesterol level from 350 mg/ml to 80 mg/ml.On review of her echocardiogram the leaflets and stent of the implantedbioprosthetic valve have normal hemodynamics over a 25 year period witha mean gradient of 10 mm Hg and an Aortic Valve area of 2.0 cm2. Thebioprosthetic valve leaflets and stent do not exhibit any evidence ofcalcification as diagnosed by Computed tomography and echocardiography.

Although the present invention has been described with reference tovarious aspects and embodiments, those of ordinary skill in the art willrecognize that changes may be made in form and detail without departingfrom the spirit and scope of the invention.

I claim: 1-16. (canceled)
 17. A method for inhibiting stenosis,obstruction and/or calcification of a mechanical heart valve followingimplantation in a vessel having a wall, said method comprising:providing a mechanical heart valve having a sewing ring covered by aGortex material; coating said mechanical heart valve, said Gortexmaterial, or both with a coating composition comprising one or moretherapeutic agents; implanting said mechanical heart valve into saidvessel in the site of a diseased natural valve site; eluting saidcoating composition from said mechanical heart valve, said Gortexmaterial or both; inhibiting stenosis, obstruction and/or calcificationof the mechanical heart valve by preventing the attachment of stem cellsto the mechanical heart valve or Gortex material, said stem cellscirculating external and proximate to the mechanical heart valve byactivating nitric oxide production (i) in the circulating stem cells,(ii) in an endothelial cell lining covering the mechanical heart valveand/or Gortex material, (iii) or both.
 18. A method for inhibitingstenosis, obstruction and/or calcification of a heart valve followingimplantation in a vessel having a wall, said method comprising:providing an unstented surgical heart valve having a sewing ringcomprising, said surgical heart valve including tissue having one ormore cusps, said surgical heart valve for replacement of a naturaldiseased valve; treating said surgical heart valve with a tissuefixative; coating said surgical heart valve, one or more cusps and/orsewing ring with a coating composition comprising one or moretherapeutic agents; implanting said surgical heart valve into saidvessel in a diseased natural valve site; eluting said coatingcomposition from said sewing ring, one or more cusps, or both;inhibiting stenosis, obstruction and/or calcification of the surgicalheart valve by preventing the attachment of stem cells to the surgicalheart valve, said stem cells circulating external and proximate to thesurgical heart valve by activating nitric oxide production (i) in thecirculating stem cells, (ii) in an endothelial cell lining covering thesurgical heart valve, (iii) or both.
 19. An apparatus for inhibitingstenosis, obstruction and/or calcification of a heart valve followingimplantation in a vessel having a wall comprising: a bioprosthetic heartvalve having one or more cusps, said bioprosthetic heart valve mountedon an elastical stent for replacement of a natural diseased valve; afirst coating composition coated on said stent, said one or more cuspsor both, said coating composition comprising one or more therapeuticagents, wherein said coating composition when eluted inhibits stenosis,obstruction and/or calcification of the bioprosthetic heart valve whenimplanted by preventing the attachment of cKit positive stem cells, SCA1cells, COP cells, and/or mesenchymal stem cells to the bioprostheticheart valve by activating nitric oxide production (i) in the circulatingstem cells, (ii) in an endothelial cell lining covering thebioprosthetic heart valve tissue, (iii) or both.
 20. The apparatus ofclaim 1 further comprising a second coating composition comprising aneNOS activator selected from Atorvastatin, Rosuvastatin, Pravastatin,Mevastatin, Fluvastatin, Simvastatin, Lovastatin, L-Arginine,citrulline, NADPH, acetylcholine, histamine, arginine vasopressin,norepinephrine, epinephrine, bradykinin, adenosine di,triphosphate,5-Hydroxytrptamine, thrombin, insulin, glucocorticoids, salicylates,L-NMMA, L-NAME, nitroglycerine, isosorbide dinitrate, isosorbide5-mononitrate, amyl nitrite, nicorandil, tetrahydrobiopterin, Zetia andcombinations of the foregoing.
 21. The apparatus of claim 1 wherein saidfirst coating composition includes one or more of (i) ananti-proliferative agent; (ii) an inhibitor of extracellular production;(iii) an inhibitor of osteoblastogenesis; and (iv) combinations of theforegoing that inhibits bone formation in an osteoblast cell originatingfrom said stem cells.
 22. The aparatus of claim 21 wherein saidanti-proliferative agent is selected from paclitaxel, sirolimus,biolimus, everolimus and combinations of the foregoing.
 23. The methodof claim 21 wherein said inhibitor of extracellular production isselected from an anti-famysltransferase inhibitor, ananti-palmitoylation inhibitor or both.
 24. The apparatus of claim 23wherein said anti-farnysltransferase inhibitor and saidanti-palmitoylation inhibitor are selected from Lonafarnib, ZarnestraR115777, FTI SCH66336, STI571,FLT-3 Inhibitor, Proteasome Inhibitor,MAPK Inhibitor, BMS-214662, Type I non-lipid inhibitors of proteinpalmitoylation, farnesyl-peptide palmitoylation or Type 2 inhibitorsmyristoyl-peptide palmitoylation, palmitoylation acyltransferaseinhibitors, lipid based palmitoylation inhibitors including2-bromopalmitoyl (2BP), tunicamycin, cerulnin and combinations of theforegoing.
 25. The apparatus of claim 21 wherein said inhibitor ofosteoblastogenesis is selected from anti-osteoporotic agents such asbisphosphonate drugs including Alendronate, Risedronate, Zoledronicacid, Etidronate, Ibandronate, Pamidronate, Tiludronate, Denosumabantibody, Calcitonin-Calcimare, Miacalcin, Forteo teriparatide,raloxfine (Evista) and combinations of the foregoing.